Methods of detecting cancer based on osteopontin

ABSTRACT

The present invention is directed to diagnostic methods based upon the expression of the protein prostasin. In particular, it is concerned with assays performed on women to determine their risk of ovarian cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. Ser. No. 09/948, 094, (nowU.S Pat. No 6,846,642), which was filed on Sep. 7, 2001. The '094application claims the benefit of U.S. provisional application No.60/231,166, filed on Sep. 7, 2000 (now abandoned).

FIELD OF THE INVENTION

The present invention is in the field of tumor cell markers and isparticularly concerned with methods of detecting cancer by assayingsamples for prostasin. In its most preferred embodiment, the inventionis directed to methods in which serum or plasma samples obtained from awoman are assayed to assess her risk of developing ovarian cancer.

BACKGROUND OF THE INVENTION

Ovarian cancer is the fifth leading cause of death from cancer in U.S.women. In most instances, a diagnosis is not made until the cancer is inan advanced state; at a time when the five year survival rate ofpatients is only about 28% (Ries, et al., SEERC Cancer Stat. Rev.1973–1995 (1998)). In contrast, the five year survival rate for womendiagnosed with localized disease is about 95%. These statistics providean incentive to search for tests that can be used to identify ovariancancer at an early stage.

The protein prostasin was originally isolated from human seminal fluidand is present at high levels in the prostate gland (Yu, et al., J.Biol. Chem. 270:13483–13489 (1995); Heid, et al., Genome Res. 6:986–994(1996)). It is expressed to a lesser extent in the kidney, liver,pancreas, salivary gland, lung and colon (Yu, et al.) J. Biol.270:13483–13489 (1995); Yu, et al., J. Biol. Chem. 269:18843–18848(1994)). Its function in these tissues has not yet been determined and aclear association between prostasin and cancer has not been established.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that prostasin can beused as a marker for identifying patients who have, or are likely todevelop, certain types of cancer. In particular, it has been found thatprostasin is elevated in the serum of women with ovarian cancer.

In its first aspect, the invention is directed to a method ofdetermining whether a human subject has, or is likely to develop, amalignant growth. A biological sample is obtained from the subject andthen assayed to determine the concentration of prostasin protein or mRNApresent. The results obtained from this assay are then compared with theresults obtained using one or more comparable biological controlsamples. In general, control samples will be of the same type as testsamples but will be obtained from a population not believed to have amalignant growth. Alternatively, controls may simply be the normalconcentration range of prostasin in comparable samples from the generalpopulation. Once assays have been completed, a conclusion may be drawnthat the subject being examined has or is likely to develop a malignantgrowth if the prostasin concentration present in the test sample issignificantly higher than in the control sample. As used herein, theterm “significantly higher” means that a difference meets the criteriafor significance accepted in the art using standard statistical methods.A significantly higher risk in this context means a probability ofhaving cancer which is greater than that of the population as a wholeand which warrants further diagnostic testing.

In general, biological samples will be samples of either serum or plasmaand prostasin levels will be determined using an ELISA type assay. Anexample of a specific assay that can be used is set forth in theExamples section below. Most preferably, tests are performed on womenfor the purpose of determining whether they are at increased risk ofhaving ovarian cancer. It is expected however that the assay will alsobe applied to other types of cancers such as cancer of the breast,prostate, lung, colon and pancreas. In another embodiment, assays areperformed using tissue samples obtained by biopsy. The prostasin levelspresent in the tissue may be evaluated by ELISA or prostasin mRNA levelsmay be determined by reverse transcription PCR.

In a more specific aspect, the invention is directed to a method ofdetermining the likelihood that a woman has or is likely to developovarian cancer by obtaining a test sample of plasma or serum from thewoman and determining the concentration of prostasin present in thesample. The results obtained are compared with results from a controlsample and a conclusion is drawn that the woman has or is likely todevelop ovarian cancer if the concentration determined for the testsample is significantly higher than the concentration in the controls.Most preferably, the test will be performed using the ELSA discussedabove. This same method can also be carried out by assaying a testsample of serum or plasma and concluding that an increased likelihood ofovarian cancer exists if the concentration of prostasin is greater than10 micrograms per ml. or, in a more stringent test, if the concentrationis greater than 12 micrograms per ml.

The assays of prostasin discussed above may either be used alone or inconjunction with other diagnostic tests. In particular, the assays maybe used in conjunction with other methods in which the concentration ofa tumor marker is determined. Such markers may include, for example,prostate specific antigen, CEA, alpha-fetoprotein and, in the case oftests for ovarian cancer, CA 125.

DETAILED DESCRIPTION OF THE INVENTION

The discovery of a correlation between prostasin concentration andovarian cancer is an outgrowth of more extensive studies on genes thatare either over- or underexpressed in cancerous ovarian epithelialcells. Numerous genes were identified which may also be used as markersand which are listed in the tables found in the Examples section below.Nevertheless, the strongest correlation has been found for prostasinwhich also has the advantage of being detectable in biological samplesthat can be readily obtained, i.e., samples of plasma or serum. Theother genes that have been correlated with the transformation of ovariancells have all been described in the literature and methods are knownfor assaying the concentration of each.

In the case of prostasin, the full length amino acid and nucleotidesequence of the human protein and gene are known in the art. These maybe found as GenBank accession number L41351. The full length cDNA is1834 nucleotides long, with the coding sequence running from nucleotide229 to 1260. The sequence is also disclosed herein as SEQ ID NO:1 (DNAsequence) and SEQ ID NO:2, amino acid sequence). The protein may bepurified using procedures described in the art or, alternatively, it canbe chemically synthesized.

Assays for prostasin expression may be determined by RT-PCR usingprimers selected from the known gene sequence. Specific examples ofprimers that can be used for successfully carrying out RT-PCR aredescribed in detail in the Examples section. ELISA assays are alsodisclosed in which the antibody described by Yu, et al. is used.However, other types of immunoassays can also be successfully used andantibodies may be produced using standard methods.

Antibodies that bind specifically to prostasin are defined for thepurposes of the present invention as those that have at least a 100 foldgreater affinity for prostasin then for any other similar undenaturedprotein. The process for producing such antibodies may involve eitherinjecting the prostasin protein itself into an appropriate animal orinjecting short peptides made to correspond to different regions ofprostasin. The peptides injected should be a minimum of 5 amino acids inlength and should be selected from regions believed to be unique to theprotein. Methods for making and detecting antibodies are well known tothose of skill in the art has evidenced by standard reference works suchas: Harlow, et al., Antibodies, Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1988); Klein, Immunology: The Science of Self-NonselfDiscrimination (1982); Kennett, et al., Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses (1980); and Campbell,“Monoclonal Antibody Technology,” in: Laboratory Techniques inBiochemistry and Molecular Biology (1984).

“Antibody,” as used herein is meant to include intact molecules as wellas fragments which retained the ability to bind antigen (e.g., Fab andF(ab′)₂ fragments). These fragments are typically produced byproteolytically cleaving intact antibodies using enzymes such as apapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). The term “antibody” also refers to both monoclonalantibodies and polyclonal antibodies. Polyclonal antibodies are derivedfrom the sera of animals immunized with the antigen. Monoclonalantibodies can be prepared using hybridoma technology (Kohler, et al.,Nature 256:495 (1975). In general, this technology involves immunizingan animal, usually a mouse, with either intact prostasin or a fragmentderived from prostasin. The splenocytes of the immunized animals areextracted and fused with suitable myeloma cells, e.g. SP₂O cells. Afterfusion, the resulting hybridoma cells are selectively maintained in HATmedium and then cloned by limiting dilution (Wands, et al.,Gastroenterology 80:225–232 (1981)). The cells obtained through suchselection are then assayed to identify clones which secrete antibodiescapable of binding to prostasin.

The antibodies or fragments of antibodies of the present invention maybe used to detect to the presence of the prostasin protein in any of avariety of immunoassays. For example, antibodies may be used inradioimmunoassays or immunometric assays, also known as “two-site” or“sandwich assays” (see Chard, “Introduction to Radioimmune Assay andRelated Techniques,” in: Laboratory Techniques in Biochemistry andMolecular Biology, North Holland Publishing Co., N.Y. (1978)). In atypical immunometric assay, a quantity of unlabelled antibody is boundto a solid support that is insoluble in the fluid being tested, e.g.,blood lymph, cellular extracts, etc. After the initial binding ofantigen to immobilized antibody, a quantity of detectably labeled secondantibody (which may or may not be the same as the first) is added topermit the detection and/or quantitation of bound antigen (see e.g.,Radioimmune Assay Method, Kirkham, et al., ed. pp. 199–206, E&SLivingston, Edinburgh (1970)). Many variations of these types of assaysare known in the art and may be employed for the detection of prostasin.

If desired, antibodies to prostasin may also be used in the purificationof the protein (see generally, Dean, et al., Affinity Chromatography, APractical Approach, IRL Press (1986)). Typically, antibody isimmobilized on a chromatographic matrix such as Sepharose 4B. The matrixis then packed into a column and the preparation containing prostasin ispassed through under conditions that promote binding, e.g., underconditions of low salt. The column is then washed and bound prostasin iseluted using a buffer that promotes disassociation of antibody, e.g., abuffer having an altered pH or salt concentration. The eluted prostasinmay be transferred into a buffer of choice, e.g., by dialysis, andeither stored or used directly.

The same basic techniques described above in connection with prostasinmay also be adapted to any of the other genes or proteins identifiedherein as being associated with ovarian cancer. Also, it should bereadily apparent that the same assays may be used in connection withother types of cancer as well, especially those of the pancreas,prostate, kidney, and lungs.

EXAMPLES Example 1 Differentially Exposed Genes from Ovarian CancerCells

A. Materials and Methods

Cell Culture

Cultures of normal human ovarian surface epithelial cells (HOSE) wereestablished by scraping the HOSE cells from the ovary and growing themin a mixture of Medium 199 and MCDB105 supplemented with 10% fetal calfserum (Mok, et al., Gynecol. Oncol. 52:247–52, (1994)). The seven HOSEcells used were HOSE17, HOSE636, HOSE642, HOSE695, HOSE697, HOSE713, andHOSE726. Ovarian cancer cell lines used were OVCA3, OVCA420, OVCA432,OVCA433, OVCA633, SKOV3, and ALST.

Microarray Probe and Hybridization

MICROMAX™ human cDNA microarray system I (NEN Life Science Products,Inc., Boston, Mass.), which contains 2400 known human cDNAs on a 1×3″slide, was used in this study. Microarray probe and hybridization wereperformed as described in the instruction manual. In brief,biotin-labeled cDNA was generated from 3 μg total RNA, which was pooledfrom HOSE17,HOSE636 and HOSE642. Dinitrophenyl (DNP)-labeled cDNA wasgenerated from 3 μg total RNA that was pooled from ovarian cancer celllines OVCA420, OVCA433 and SKOV3. Before the cDNA reaction, an equalamount of RNA control was added to each batch of the RNA samples fornormalization during data analysis. Biotin-labeled and DNP-labeled cDNAwere mixed, dried and resuspended in 20 μl hybridization buffer, whichwas added to the cDNA microarray and covered with a coverslip.Hybridization was carried out overnight at 65° C. inside a hybridizationcassette.

Post hybridization and Cyanine-3 (Cy3™) and Cyanine-5 (Cy5™) TyramideSignal Amplification (TSA)

After hybridization, microarrays were washed with 30 ml 0.5×SSC, 0.01%SDS, and then 30 ml 0.06×SSC, 0.01% SDS. Finally the microarray waswashed with 0.06×SSC. Hybridization signal from biotin-labeled cDNA wasamplified with streptavidin-horseradish peroxidase and Cy5™-tyramide,while hybridization signal from DNP-labeled cDNA was amplified withanti-DNP-horseradish peroxidase and Cy3™-tyramide. After signalamplification and post-hybridization wash, cDNA microarray was air-driedand detected with a laser scanner.

Image Acquisition and Data Analysis

Cy3 signal was derived from ovarian cancer cells and Cy5 signal wasderived from HOSE cells. Laser detection of the Cy3 and Cy5 signal onthe microarray was acquired with a confocal laser reader, ScanArray3000(GSI Lumonics, Watertown, Mass.). Separate scans were taken for eachfluor at a pixel size of 10μ. cDNA derived from the control RNAhybridized to 12 specific spots within the microarray. Cy3 and Cy5signals from these 12 spots should theoretically be equal and were usedto normalize the different efficiencies in labeling and detection withthe two fluors. The fluorescence signal intensities and the Cy3/Cy5ratios for each of the 2400 cDNAs were analyzed by the software Imagene3.0 (Biodiscovery Inc, Los Angeles, Calif.).

Real-Time Quantitative RT-PCR

Real-time PCR was performed in duplicate using primer sets specific toGA733-2, osteopontin, prostasin, creatine kinase B, CEA, KOC and ahousekeeping gene, cyclosporin, in an ABI PRISM 5700 Sequence Detector.RNA was first extracted form normal ovarian epithelial cell cultures(HOSE695, 697, 713, and 726) and six ovarian carcinoma cell lines(OVCA3, OVCA432, OVCA433, OVCA633, SKOV3 and ALST). cDNA were generatedfrom 1 μg total RNA using the TaqMan reverse transcription reagentscontaining 1× TaqMan RT buffer, 5.5 mM MgCl₂, 500 μM dNTP, 2.5 μM randomhexamer, 0.4 U/μl RNase inhibitor, 1.25 U/μl MultiScribe reversetranscriptase (PE Applied Biosystems, Foster City, Calif.) in 100 μl.The reaction was incubated at 25° C. for 10 min, 48° C. for 30 min andfinally at 95° C. for 5 min. 0.5 μl of cDNA was used in a 20 μl PCR mixcontaining 1×SYBR PCR buffer, 3 mM MgCl₂, 0.8 mM dNTP, and 0.025 U/μlAmpliTaq Gold (PE Applied Biosystems, Foster City, Calif.).Amplification was then performed with denaturation for 10 min at 95° C.,followed by 40 PCR cycles of denaturation at 95° C. for 15 sec andannealing/extension at 60° C. for 1 min. The changes in fluorescence ofSYBR Green I dye in every cycle was monitored by the ABI5700 systemsoftware, and the threshold cycle (C_(T)) for each reaction wascalculated. The relative amount of PCR products generated from eachprimer set was determined based on the threshold-cycle or C_(T) value.Cyclosporin was used for the normalization of quantity of RNA used. ItsC_(T) value was then subtracted from that of each target gene to obtaina ΔCT value. The difference (ΔΔC_(T)) between the ΔC_(T) values of thesamples for each gene target and the ΔC_(T) value of the calibrator(HOSE726) was determined. The relative quantitative value was expressedas 2^(−ΔΔCT).

B. Results and Discussion

The MICROMAX System

The MICROMAX system allows the simultaneous analysis of the expressionlevel of 2400 known genes. The use of TSA signal amplification in thesystem after hybridization reduces the amount of total RNA needed to afew micrograms which is about 20–100 times less than currently usedmethods. The details of TSA have been described previously forchromosome mapping of PCR-labeled probes less than 1 kb by FISH(Schriml, et al., Biotechniques 27:608–611, (1999)). In this study, 30putative differentially over-expressed genes (excluding 9 ribosomalgenes) were identified in ovarian cancer cell lines (Table 1). Usinghigh density cDNA array on membranes, Schummer et al. (Schummer, et al.,Gene 238:375–385, (1999)) has identified 32 known genes that exhibit atumor-to-HOSE ratios of more than 2.5-fold. Fourteen of these 32 geneswere present in the MICROMAX cDNA microarray but only five of them werepresent at more than 3-fold.

Biotin-labeled cDNA was made from ovarian cancer cell lines, whileDNP-labeled cDNA was made from HOSE cells. The differential TSAamplification of the hybridization signal depends on the use of aSteptavidin-HRP conjugate or anti-DNP-HRP conjugate in a sequentialstep. At each step, cyanine-5-Tyramide or cyanine-3-Tyramide can beadded and the HRP will then catalyze the deposit of Cy3 or Cy5 onto thehybridized cDNA nonspecifically. As a result, either Cy3 or Cy5 signalscan be used for the cDNA derived from ovarian cancer cell lines, andvice versa for HOSE cells. Thus, it is not necessary to make twodifferent sets of probes to compare the effect of Cy3 or Cy5fluorescence as a result of their differences in extinction coefficientsand quantum yields. Cy3 and Cy5 signals on the processed slides werestable for more than 6 months.

Normalization of Signals

The MICROMAX system has 3 nonhuman genes as internal controls. Each ofthe control genes is spotted 4 times on the microarray. Equal amounts ofpolyA RNA derived from these control genes were spiked into the totalRNA samples derived from both HOSE and ovarian cancer cell lines duringcDNA synthesis. Thus, hybridization signals from these control genes intwo RNA samples should theoretically be the same. The Cy3 to Cy5 ratiosfor these control genes varied from 0.4 to 4.0 and the average ratio was1.5±1.1. From a prior microarray analysis of human cancer cells, 88genes have been identified to express at relatively constant levels inmany cell types (DeRisi, et al., Nat. Genet. 14:457–60, (1996)). TheMICROMAX microarray also contains 58 of these 88 genes and 21 of thesegenes with signal to background ratio more than 3-fold were analyzed(Table 2). The ratios varied from 0.23 to 5.22. The average ratio is1.6±1.5. Thus, the result of internal control RNA for normalizing signalwas similar to that of genes that express at a relatively constant levelin different cell types.

Effect of Background Signal on the Identification of DifferentiallyExpressed Genes

In the present study, 1357 of the 2400 genes on the microarray have Cy3signals (from ovarian cancer cell lines) that were at least two-foldhigher than the background, and 740 genes have Cy3 signals that were atleast three-fold higher than the background. After post-hybridizationwashes, there was still significant background intensity for the Cy3signal but very low background for Cy5. Subsequently, the microarray waswashed again in 30 ml TNT buffer at 42° C. for 20 minutes instead of atroom temperature, followed by 30 ml of 0.006×SSC for 1 minute. Thewashed microarray was then dried and re-scanned. This process wasrepeated several times until the number of genes with signal tobackground ratios at least 3-fold remained the same. The extensivewashing steps decreased the background intensity significantly, butthere was no obvious changes in the signal intensity. As a result, thenumber of genes with at least 3-fold signal to background ratiosincreased from 740 to 791 genes. Moreover, the differential expressionratios, in general, also increased (Table 1 to Table 2). Moreimportantly, after the extensive washing, we were able to detect thedifferential expression of two weakly expressed genes, thiol-specificantioxidant protein (4.5-fold) and elongation factor-1-β (9.7-fold),which were previously identified by Schummer et al. Thus, the extensivepost-hybridization washing and re-scanning of signals may be necessaryto decrease background signal especially in the case of differentiallyexpressed genes with low expression levels.

Confirmation of Differential Expression by Real-Time Quantitative PCR

To further validate differential expression, five interesting genes werechosen, GA733-2, osteopontin, koc, prostasin, and creatine kinase B, forreal-time PCR analysis. All these genes are either surface antigens orsecreted proteins. Thus, they may be useful as tumor markers for ovariancancer. GA733-2 is a cell surface 40-kDa glycoprotein associated withhuman carcinomas of various origins (Szala, et al., Proc. Natl. Acad.Sci. USA 87:3542–6, (1990)). Osteopontin is a secreted glycoprotein witha conserved Arg-Gly-Asp (RGD) integrin-binding motif and is expressedpredominantly in bone, but has also been found in breast cancer andthyroid carcinoma with enhanced invasiveness (Sharp, et al., Lab.Investigat. 79:869–877, (1999), Tuck, et al., Oncogene 18:4237–4246,(1999)). Prostasin is a novel secreted serine proteinase which wasoriginally identified in seminal fluid (Yu, et al., J. Biol. Chem.269:18843–8, (1994)). The koc transcript is highly over-expressed inpancreatic cancer cell lines as well as in pancreatic cancer. It isspeculated that koc may assume a role in the regulation of tumor cellproliferation by interfering with transcriptional and orposttranscriptional processes (Mueller-Pillasch, et al., Oncogene14:2729–33, (1997)). Creatine kinase B is a serum marker associated withrenal carcinoma and lung cancer (Kurtz, et al., Cancer 56:562–6, (1985),Takashi, et al., Urologia Internationalis 48:144–8, (1992)). Tworandomly selected genes, CEA and RGS, were used as negative controls.

The results showed that all the tested ovarian cancer cell linesexpressed higher levels of GA733-2. However, osteopontin, prostasin, KOCand creatine kinase B were over-expressed in only some of the cancercell lines. Since we were using pools of RNA, the differentialexpression that was observed is an average of the gene expression from 3independent HOSE cells or 3 different cancer cell lines. This strategyallows us to capture genes that overexpress in either some or all of thecell lines. Genes that only overexpress in some of the ovarian cancercell lines may be useful for molecular classification of ovarian cancercells. Since as little as 10 pg cDNA is enough for real-timequantitative RT-PCR reaction, RNA extracted from microdissected tissuewould be enough for thousands of such real-time quantitative RT PCRanalyses.

TABLE 1 List of genes differentially over-expressed in ovarian cancercells more than 10-fold. Before After extensive extensive washingwashing Cy3 signal Accession # Description (Cy3/Cy5) (Cy3/Cy5) intensityM33011 carcinoma-associated antigen 472 444 1249 GA733-2 J04765Osteopontin 156 184 11851 L41351 Prostasin 44 170 3172 L19783 GPI-H 4 88916 U96759 Von Hippel-Lindau binding protein 60 59 1377 (VBP-1) M57730B61 20 49 5514 L33930 CD24 signal transducer and 3′ 24 47 26722 regionD55672 hnRNP D 45 44 950 U97188 Putative RNA binding protein KOC 223 383599 L19871 ATF3 9 37 3507 J04991 p18 15 34 9914 D00762 mRNA forproteasome subunit HC8 17 29 4703 U17989 Nuclear autoantigen GS2NA 5 28721 U43148 Patched homolog (PTC) 10 28 4155 AF010312 Pig7(PIG7) 13 2317379 M80244 E16 18 21 4180 X99802 mRNA for ZYG homologue 14 21 2086U05598 Dihydrodioldehydrogenase 10 18 21595 L47647 Creatine kinase B. 718 787 M55284 Protein kinase C-L (PRKCL) 7 16 863 X15722 mRNA forglutathione reductase 23 14 794 554005 Thymosin beta-10 6 13 1476AB006965 mRNA for Dnmlp/Vpslp-like 7 13 4183 protein M83653 Cytoplasmicphosphotyrosyl protein 6 13 2156 Phosphatase X12597 mRNA for highmobility group-1 7 12 2785 protein (HMG-1) M18112 poly(ADP-ribose)polymerase 6 12 9277 U56816 Kinase Mytl (Mytl) 4 11 1773 X06233 mRNA forcalcium-binding protein 7 11 3007 In macrophages (MRP-14) D85181 mRNAfor fungal sterol-C5- 6 11 3571 desaturase homolog M31627 X box bindingprotein-1 (XBP-1) 5 10 12151

TABLE 2 Cy3 versus Cy5 ratio for a set of genes that are previouslyshown to express at relative constant level (2) Before After extensiveextensive Accession washing washing # Description (Cy3/Cy5) (Cy3/Cy5)X06323 MRL3 mRNA for ribosomal 3.31 5.22 protein L3 homologue AF0060433-phosphoglycerate dehydrogenase 3.81 4.8 M37400 Cytosolic aspartateaminotransferase 3.03 3.66 D30655 mRNA for eukaryotic initiation factor4.17 3.48 4A11 J04208 inosine-5′-monophosphate 1.13 2.15 dehydrogenase(IMP) M17885 Acidic ribosomal phosphoprotein PO 2.74 2.09 X54326 mRNAfor glutaminyl-tRNA 1.17 2.01 synthetase J04973 Cytochrome bc-1 complexcore 0.98 1.6 protein II D13900 mitochondrial short-chain. enoyl-CoA0.91 1.52 hydratase Z1531 mRNA for elongation factor 0.76 0.89 1-gammaD78361 mRNA for ornithine decarboxylase 0.51 0.82 antizyme U13261 e1F-2associated p67 homolog 0.41 0.82 X15183 mRNA for 90-kDa heat-shockprotein 0.8 0.75 M36340 ADP-ribosylation factor 1 (ARF1) 0.5 0.66 X91257mRNA for seryl-tRNA synthetase 0.75 0.52 AF047470 Malate dehydrogenaseprecursor 0.41 0.51 (MDH) mRNA D13748 mRNA for eukaryotic initiationfactor 0.33 0.43 4A1 L36151 Phosphatidylinosito14-kinase 0.26 0.38X04297 mRNA for Na, K-ATPase 0.27 0.34 alpha-subunit X79535 mRNA forbeta tubulin, clone 0.18 0.28 nuk_278 J04173 Phosphogylcerate mutase(PGAM-B) 0.14 0.23

TABLE 3 Real-time quantitative RT-PCR analysis of a few selectedgenes^(a). Creatin GA733-2 Osteopontin KOC Prostasin Kinase B CEA RGSNormal ovarian cells HOSE695 4 21 5 28 0.4 5 32 HOSE697 1 1 2 4 0.4 3 18HOSE713 1 20 7 5 1 16 25 HOSE726 1 1 1 1 1 1 1 Average (HOSE) 2 11 4 9 16 19 Ovarian cancer cell lines OVCA3 419 6 4 61 393 1 4 OVCA432 136 0 017 8 0 1 OVAC433 2048 0 52 57 12 1 4 OVCA633 2917 13777 3 228 4 15 27SKOV3 2856 265 10 2 31 6 13 ALST 3875 6081 78 10 1 2 5 Average 2042 335524 62 75 4 9 (OVCA) OVCA/HOSE 1361 310 6 7 103 1 0.5 (average) ^(a)Eachgene was analyzed using an identical panel of 10 cDNA samples thatcomprised of 4 normal ovarian surface epithelial cells and 6 ovariancancer cell lines. The expression of each gene for each cDNA sample wasnormalized against cyclosporin. Duplicated reactions were performed foreach of the genes and similar results were obtained.

Example 2 Differentially Expressed Genes

A. Choice of Samples and the Identification of Differentially ExpressedGenes

We compared the expression of 2400 genes between primary human ovariansurface epithelial (HOSE) cells and ovarian cancer (OVCA) cells usingthe MICROMAX™ cDNA microarray system (NEN Life Science Products, Boston,Mass., USA). Three primary HOSE cells from different individuals werepooled together as a normal sample. The use of pooled normal samples hastwo advantages—(1) fluctuations in gene expression among normal HOSEcells due to the individual difference in age or physiological statesmay be minimized, and (2) a sufficient amount of RNA for directlabelling can be obtained from the precious primary cell cultures.Similarly, three different cancer cell lines were pooled together as anovarian cancer sample for the analysis.

47 genes over-expressed (Table 4 and Table 5), and 58 genesdown-regulated in ovarian cancer cells (Table 6) were identified from asingle microarry experiment. However, the list of genes described hereis different from two similar studies reported previously (Schummer, etal., Gene 238:375 (1999), Wang, et al., Gene 229:101, (1999)). Only afew differentially expressed genes were shared by these studies. Thedifferences in the list of differentially expressed genes may be due tothe use of different samples in the analysis. We compared the geneexpression of primary normal ovarian surface epithelial cells andovarian cancer cell lines. In one of the previous studies, geneexpression of normal ovary was compared with tumor tissues, while geneexpression of low passage, ovarian surface epithelial cells werecompared with tumor tissues in another study. Apparently, the choice ofsamples for analysis would account for the different set of genesidentified.

B. Background Hybridization Signal and the Identification of WeaklyExpressed Genes

Gene expression from OVCA samples was detected as a Cy3 signal whilegene expression from HOSE samples was detected as Cy5 signal. Aftercompletion of the recommended procedures, significant background signalwas still observed in the Cy3 signal that derived from OVCA sample. Aseries of additional, stringent post-hybridization washes reduced thebackground signal. While the Cy3 to Cy5 ratios for most of the genesincreased slightly after stringent post-hybridization washes, theratios, for some genes increased significantly. More importantly, afterthe stringent post-hybridization washes, we were able to detect theweakly expressed genes that are differentially over-expressed in ovariancancer cells (Table 5).

C. Genes Over-Expressed in Ovarian Cancer Cells

From the list of over-expressed genes, several putative mechanisms maybe involved in the pathogenesis of ovarian cancer—(1) inactivation of atumor suppressor, (2) altered expression of transcription factors, (3)overexpression of oncogenes, (4) overexpression ofglycosylphosphatidylinositol (GPI) anchor associated proteins, and (5)altered cell cycle control. According to this list of genes, VBP1interacts with the product of the von Hippel-Lindau gene and is expectedto participate in pathways by inactivation of this tumor suppressorgene. RNA binding proteins, Koc and hnRNP D, may assume a role in theregulation of tumor cell proliferation by interfering withtranscriptional and/or posttranscriptional processes of tumor suppressorgenes. However, the precise role of these RNA binding proteins in humantumor cells remains to be elucidated. ATF3 and XBP-1 are transcriptionfactors which may play an important role in the regulation of geneexpression by cAMP-dependent intracellular signaling pathways and beessential for hepatocyte growth respectively. Also related to genetranscription, HMG-I protein has been implicated as a potential markerfor thyroid carcinoma. p18 and E16 are two oncogenes that have beenfound to be over-expressed in acute leukemia cells and various humancancers respectively. The glycosylphosphatidylinositol (GPI) anchor,potentially capable of generating a number of second messengers, such asdiacylglycerol, phosphatidic acid, and inositol phosphate glycan, hasbeen postulated to be involved in signal transduction in various celltypes, including T-cells. Genes encoding GPI anchored proteins. (GPI-H,B61 and CD24) were found to be over-expressed in ovarian cancer cells.Mytl activity is temporally regulated during the cell cycle and issuggested to play a role in mitotic control. CD24, a GPI anchoredprotein, is also involved in cell cycle control.

The differential expression of five interesting genes, GA733-2,osteopontin, koc; prostasin, and creatine kinase B, has previously beenconfirmed by real-time RT-PCR. All these genes are either surfaceantigens or secreted proteins, which may be potential serum markers. Infact, we have found that prostasin is significantly higher in the plasmaof an ovarian cancer patient. GA733-2 is known as epithelial cellsurface antigen (EPG) or adenocarcinoma-associated antigen (KSA). Theseproteins may function as growth factor receptors. Osteopontin is anacidic phosphorylated glycoprotein of about 40 Kd which is abundant inthe mineral matrix of bones and possibly functions as a cell attachmentfactor involved in tumor invasion and metastasis. Prostasin is a serineproteinase expressed in prostate and prostate carcinoma. Creatine kinasehas been shown to be at an elevated level in the blood of patients withrenal cell carcinoma or small lung carcinoma.

D. Genes Down-Regulated in Ovarian Cancer Cells

More than 50 genes down-regulated in ovarian cancer cells wereidentified in this study. In this list of genes (Table 6),SPARC/osteonectin has been previously identified as a down-regulatedgene. SPARC is an extracellular matrix (ECM) protein withtumor-suppressing activity in human ovarian epithelial cells (Mok, etal., Oncogene 12:1895, (1996)). Other ECM or ECM related proteins suchas fibronectin, tenascin, OB-cadherin-1, HXB, matrix metalloproteinase,and ICAM-1 were also found to be down-regulated. Tenascin has beensuggested to be a prognostic marker for colon cancer. Patients with moretenascin expression have better long-term survival than patients with noor weak expression.

Several other genes involved in responding to growth factors or mitogenswere also down-regulated. These genes were Shps-1, phosphorylase-kinase,phosphoinositide 3-kinase, NDP kinase, ZIP-kinase, signal transducingguanine nucleotide-binding regulatory protein, IGFBP2, TGF-beta, andTNFα receptor. SHPS-1, a novel glycoprotein, binds theSh2-domain-containing protein tyrosine phosphatase SHP-2 in response tomitogens and cell adhesion. Suppression of SHPS-1 expression by v-Srcvia the Ras-MAP kinase pathway has been shown to promote the oncogenicgrowth of cells. NDP kinase gene located on chromosome 17q has beenproposed as a metastasis suppressor gene in a variety of tumor types.ZIP kinase is a novel serine/threonine kinase and has been shown tomediate apoptosis through its catalytic activities. Previous worksuggests that the TGF-beta receptor complex and its downstream signalingintermediates constitute a tumor suppressor pathway. The stabilizationof TNF-alpha receptors on the surface of human colon carcinoma cells isnecessary for TNFα induced cell death. Besides these two major groups ofgenes, other genes encoding proteases and complement C1 components werealso down-regulated. Some of these down-regulated genes, such astestican and osteoblast specific factor 2, have not yet been associatedwith carcinogenesis.

TABLE 4 OVCA Accession (OVCA/ signal # Description HOSE) intensityM33011 carcinoma-associated antigen 444 1249 GA733-2 704765 Osteopontin184 11851 L41351 Prostasin 170 3172 L19783 GPI-H 88 916 U96759 VonHippel-Lindau binding protein 59 1377 (VBP-1) M57730 B61 49 5514 L33930CD24 signal transducer and 3′ region 47 26722 D55672 hnRNP D 44 950U97188 Putative RNA binding protein KOC 38 3599 L19871 ATF3 37 3507704991 p18 34 9914 D00762 proteasome subunit HC8 29 4703 U17989 Nuclearautoantigen GS2NA 28 721 U43148 Patched homolog (PTC) 28 4155 AF010312Pig7 (PIG7) 23 17379 M80244 E16 21 4180 X99802 ZYG homologue 21 2086U05598 Dihydrodiol dehydrogenase 18 21595 L47647 Creatine kinase B. 18787 M55284 Protein kinase C-L (PRKCL) 16 863 X15722 glutathionereductase 14 794 554005 Thymosin beta-10 13 1476 AB006965Dnmlp/Vpslp-like protein 13 4183 M83653 Cytoplasmic phosphotyrosylprotein 13 2156 phosphatase X12597 high mobility group-1 protein 12 2785(HMG-1) M18112 poly(ADP-ribose) polymerise 12 9277 U56816 Kinase Mytl(Mytl) 11 1773 X06233 calcium-binding protein in 11 3007 macrophages(MRP-14) D85181 fungal sterol-C5-desaturase homolog 11 3571 M31627 X boxbinding protein-1 (XBP-1) 10 12151

TABLE 5 Weakly expressed genes identified after stringent washes.AF005654 Actin-binding double-zinc-finger protein 18770 751 (abLIM).L10844 Cellular growth-regulating protein. 33 725 M88163 Globaltranscription activator homologous 18 642 sequence. U02882Rolipram-sensitive 3′, 5′-cyclic AMP 27 636 phosphodiesterase. X12517 U1small nuclear RNP-specific C protein. 112 550 D29833 Salivary prolinerich peptide P-B. 10 492 AF020918 Glutathione transferase GSTA4 47 475J05262. Farnesyl pyrophosphate synthetase 95 469 L08424 Achaete scutehomologous protein 57 457 (ASH1). M84526. Adipsin/complement factor D 65441 U35113 Metastasis-associated mtal. 13 367 D28468 DNA-binding proteinTAXREB302. 268 357 AF012126 Zinc finger protein (ZNF198). 15 342ABOO0714 RVP1. 11 314 AF029750 Tapasin (NGS-17). 118 305 X60489Elongation factor-1-beta. 10 282 L36645 Receptor protein-tyrosine kinase(HEK8). 71 273

TABLE 6 List of genes down-regulated in ovarian cancer cells more than10-fold. Accession (HOSE/ HOSE # Description OVCA) signal D45421phosphodiesterase I alpha ∞ 667 M35410 Insulin-like growth factorbinding ∞ 1517 protein 2 (IGFBP2) X81334 collagenase-3 protein ∞ 5146D13665 osteoblast specific factor 2 (OSF-2pl) ∞ 24300 J03040SPARC/osteonectin ∞ 28711 D86043 SHPS-1 681  9450 U89942 Lysyloxidase-related protein (WS9-14) 454  25055 M59807 NK4 118  22438 Z74616Prepro-alpha2(I) collagen 101  2281 Z74615 Prepro-alpha1(I) collagen 8124323 X06596 Complement component Cls 76 22672 M95787 22 kDa smoothmuscle protein (SM22) 71 31581 X06256 Fibronectin receptor alpha subunit66 27901 M36981 Putative NDP kinase (nm23 H2S) 60 15411 AJ001838Maleylacetoacetate isomerase 59 304 X56160 Tenascin 42 36062 D21254OB-cadherin-1 40 20621 Y07921 Serine protease 37 2247 Y10032 Putativeserine/threonine protein kinase 36 8510 X04526 Beta-subunit signaltransducing 36 13321 proteins Gs/Gi (beta-G) L31409 Creatine transporter35 8677 X04701 Complement component Clr 33 10299 X13839 Vascular smoothmuscle alpha-actin 32 27311 X84908 Phosphorylase-kinase, beta subunit.30 11341 L14595 Alanine/serine/cysteine/threonine 27 2234 transporter(ASCTI) M97796 Helix-loop-helix protein (Id-2) 25 3187 X04741 Proteingene product (PGP) 9.5 25 13138 Y10055 Phosphoinositide 3-kinase 23 614X83535 Membrane-type matrix 20 6464 metalloproteinase U69546 RNA bindingprotein Etr-3 18 2714 U16268 AMP deaminase isoform L, 18 3512alternatively spliced (AMPD2) mRNA, exons 1B, 2 and 3. S59749 5E10antigen 18 1249 U03057 Actin bundling protein (HSN) 17 9285 J0285420-kDa myosin light chain (MLC-2) 16 8323 X16940 Enteric smooth musclegamma-actin 16 18776 X13223 N-acetylglucosamide-(beta 1-4)- 16 3357galactosyltransferase M69181 Nonmuscle myosin heavy chain-B 16 11126(MYH10) X06990 ICAM-1 16 25605 M13656 Plasma protease (C1) inhibitor I51091 X73608 Testican 15 2977 M96803 General beta-spectrin (SPTBNI) 1513359 D00632 Glutathione peroxidase 15 4951 X03445 Nuclear envelopeprotein lamin C 13 17451 precursor L06419 Lysyl hydroxylase (PLOD) 139288 M12125 Fibroblast muscle-type tropomyosin 12 34379 S45630 AlphaB-crystallin, Rosenthal fiber 12 1536 component. AB005298 BAI2 11 3839L77864 Stat-like protein (Fe65) 11 1914 AB007144 ZIP-kinase 11 4277M16538 Signal-transducing guanine nucleotide- 11 2793 binding regulatory(G) protein beta subunit L35545 Endothelial cell protein C/APC 11 1002receptor (EPCR) M75161 Granulin 11 13289 X69910 p63 11 16460 D12686eIF-4 gamma 11 21555 L07594 Transforming growth factor-beta type 11 672III receptor (TGF-beta) M33294 Tumor necrosis factor receptor 10 6592U18121 136-kDa double-stranded RNA binding 10 1619 protein p136(K88dsRBP) M55618 Hexabrachion (HXB) 10 3866

Example 3 Prostasin as a Serum Marker for Ovarian Cancer

I. Materials and Methods

Biological Specimens

Ovarian tissue and cells were freshly collected from women undergoingsurgery at the Brigham and Women's Hospital for diagnosis of primaryovarian cancer or from control subjects having a hysterectomy andophorectomy for benign disease. Cultures of normal ovarian surfaceepithelial (HOSE) cells were established by scraping the surface of theovary and growing recovered cells in a mixture of medium 199 and MCDB105 medium supplemented with 10% fetal calf serum. The following sevennormal HOSE cells were used: HOSE17, HOSE636, HOSE642, HOSE697, HOSE713,HOSE726 and HOSE730. Ovarian cell lines were established by recoveryfrom ascites fluid or explanted from solid tumors. The following tenovarian cancer cell lines were used: OVCA3, OVCA420, OVCA429, OVCA432,OVCA433, OVCA633, CAOV3, DOV13, ALST and SKOV3.

Serum specimens from women with ovarian cancer, other gynecologiccancers and benign gynecologic disorders requiring hysterectomy and fromnon-diseased normal women were obtained from discarded specimens, fromdiscarded specimens that were archived during the period from 1983through 1988 or from specimens collected under more recent protocolssince 1996. The archived samples were collected from several studiesassessing the performance of CA 125 in a variety of diagnosticcircumstances, including gynecologically normal subjects as well assubjects having exploratory surgery for pelvic masses that proved to beovarian, cervical or endometrial cancer for a benign disease such as afibroid tumor. The archived specimens were stored at −70° C. However,thawing was known to have occurred once for some of the archivedspecimens. More recent specimens were obtained within the past fiveyears and were stored at −70° C. without any incident of thawing. Inboth specimen banks, serum from case patients with ovarian cancer andserum from control patients were collected concurrently.

Microarray Probe and Hybridization

The MICROMAX™ Human cDNA Microarray System I(NEN Life Science Products,Inc. Boston, Mass.) was used in this study. Biotin-labeled cDNA wasgenerated-from 3 micrograms of total RNA that was pooled from HOSE17,HOSE636 and HOSE642 cells. Dinitrophenyl-labelled cDNA was generatedfrom 3 micrograms of total RNA that was pooled from ovarian cancer celllines OVCA420, OVCA433, and SKOV3. Before the cDNA reaction, 5 ng ofArabidopsis control RNA were added to each batch of the RNA samples forthe normalization of hybridization signals. The biotin-labelled cDNA andthe dinitrophenyl-labelled cDNA were mixed, dried and resuspended in 20microliters of hybridization buffer 5× standard saline citrate (SSC),0.1% sodium dodecyl sulfate (SDS) and salmon sperm DNA at 0.1 mg/ml(1×SSC=0.15 M NaCl, 0.15 M sodium citrate, pH 7). This mixture was addedto the cDNA microarray and was covered with a coverslip. Hybridizationwas carried out overnight at 65° C. inside a hybridization cassette.

After hybridization, the microarray was washed with 30 ml of0.5×SSC-0.01% SDS, with 30 ml of 0.06×SSC-0.01% SDS and then with 30 mlof 0.06×SSC alone. The hybridization signal from biotin-labelled cDNAwas amplified with streptavidin-horseradish peroxidase and fluorescentdye, Cy5-tyramide. The hybridization signal from thedinitrophenyl-labelled cDNA was amplified withanti-dinitrophenyl-horseradish peroxidase and another fluorescent dye,Cy3-tyramide. After signal amplification and post-hybridization wash inTNT buffer (i.e., 0.1 M Tris-HCl (pH 7.5)-0.15 M NaCl-0.15% Tween20),the microarray was air-dried and signal amplification was detected witha laser scanner.

Laser detection of the Cy3 signal (derived from ovarian cancer cells)and the Cy5 signal (derived from HOSE cells) on the microarray wasacquired with a confocal laser reader. Separate scans were taken foreach fluor at a pixel size of 10 micrometers. cDNA derived from theadded Arabidopsis RNA hybridized to 12 specific spots on the microarray,which were composed of DNA sequences obtained from four differentArabidopsis expressed sequence tags in triplicate. Cy3 and Cy5 signalsfrom these 12 spots should theoretically be equal and were used tonormalize the different efficiencies in labeling and detection with thetwo fluors. The fluorescence signal intensity and the ratio of thesignals from Cy3 and Cy5 for each of the 2400 cDNAs were analyzed by thesoftware Imagene 3.0 (Biodiscovery Inc., Los Angeles, Calif.).

Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction

Real-time reverse transcription-polymerase chain reaction (RT-PCR) wasperformed in duplicate by using primer sets specific for theoverexpressed gene encoding the secretory protein prostasin (forwardprimer=5′-ACTTGAGCCACTCCTTCCTTCAG-3′ (SEQ ID NO:3); reverseprimer=5′-CTGATGGTCCCAAAAAGCACAC-3′ (SEQ ID NO:4)) and a housekeepinggene, GADPH. RNA was first extracted from normal ovarian epithelial cellcultures (HOSE697, HOSE713, HOSE726, and HOSE730) and from 10 ovariancarcinoma cell lines (OVCA3, OVCA420, OVCA429, OVCA432, OVCA433,OVCA633, CAOV3, DOV13, SKOV3, and ALST). cDNA was generated from 1microgram of total RNA using the TaqMan RT reagents containing 1× TaqManreverse transcriptase buffer, 5.5 mM MgCl₂, all four deoxyribonucleosidetriphosphates (each at 500 μM), 2.5 μM random hexamers, MultiScribereverse transcriptase at 1.25 U/μl, and RNasin at 0.4 U/μl in 100 μl.The reaction was incubated at 25° C. for ten minutes at 48° C. forthirty minutes and finally at 95° C. for five minutes. A total of onemicrogram of cDNA was used in 20 μl PCR mixture containing 1×SYBR PCRbuffer, 3 mM MgCl₂, all for deoxyribonucleoside triphosphates (each at0.8 mM) and AmpliTaq Gold. The cDNAs were then amplified by denaturationfor ten minutes at 95° C., followed by 40 PCR cycles of denaturation at95° C. for 15 seconds and annealing-extension at 60° C. for one minute.The changes in fluorescence of the SYBR Green I dye in every cycle weremonitored by ABI 5700 system software and the threshhold cycle, whichrepresents the PCR cycle at which an increase in reporter fluorescenceabove a baseline signal can first be detected for each reaction, wascalculated. The relative amount of PCR products generated from eachprimer set was determined on the basis of the threshhold cycle (C_(T))value. GAPDH was used to normalize the quantity of RNA used. Its C_(T)value was then subtracted from each target gene to obtain a ΔC_(T)value. The difference between the ΔC_(T) values of the samples for eachgene target and the ΔC_(T) value of a calibrator which served as aphysiologic reference was determined. For confirmation of thespecificity of the PCR, PCR products were subjected to electrophoresison a 1.2% agrose gel. A single PCR product with the expected size shouldbe observed in samples that express the gene of interest.

Immunohistochemical Localization of Prostasin

Immunostaining with anti-prostasin antibody was performed on sectionsprepared from two normal ovaries, from two serous borderline ovariantumors, and from two grade 1, two grade 2, and two grade 3 serousovarian adenocarcinomas. This rapid polyclonal antibody, also used inthe serum assay was prepared from prostasin purified from human seminalfluid as described previously (Yu, et al., J. Biol. Chem.269:18843–18848 (1994)). Tissues were fixed in 4% paraformaldehyde andembedded in paraffin. Sections (5 μm) were cut, mounted on microscopicslides and incubated at 50° C. overnight. They were then transferred toTris-buffered saline (TBS) and quenched in 0.2% H₂O₂ for 20 minutes.After quenching, the sections Were washed in TBS for 20 minutes,incubated with normal horse serum for 20 minutes, and then incubatedwith anti-prostasin polyclonal antibody (diluted 1:400) at roomtemperature for one hour. The slides were then washed in TBS for 10minutes, incubated with diluted biotinylated secondary horse anti-rabbitantibody solution for 30 minutes, washed again in TBS for 10 minutes,incubated with avidin-biotin complex reagent for 30 minutes and washedin TBS for 10 minutes. Stain development was performed for 5 minutesusing a diaminobenzidine kit. Finally, the sections were washed in waterfor 10 minutes. They were then counterstained with hemanoxylin,dehydrated with an ascending series of alcohol solutions, cleared inxylene and mounted. The specificity of the staining was confirmed byusing preimmunization rabbit serum and by preabsorbing the antibody withthe purified peptide (60 mg/ml) or prostasin for 2 hours at 37° C.before applying the adsorbed antiserum to the sections.

Measurement of Prostasin and CA 125 in Sera

Sera were available from a total of 201 subjects (64 case patients withovarian cancer and 137 control subjects, including 34 with othergynecologic cancers, 42 with benign gynecologic diseases, and 71 with noknown gynecologic diseases). In all of the case patients and in the 68control subjects who had surgery, preoperative specimens were available.Serum levels of immunoreactive human prostasin were determined by theenzyme-linked immunosorbent assay (ELICA) prepared with the previouslydescribed antibody to human prostasin. Microtiter plates (96 well) werecoated with anti-prostasin immunoglobulin G (IgG) (1 μg/ml, 100 μl perwell) overnight at 4° C. Purified prostasin standards or samples wereadded to individual wells in a total volume of 100 μl ofphosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin(dilution buffer) and incubated at 37° C. for 90 minutes. Biotin labeledanti-human prostasin IgG was added to each well at a concentration of 1μg/ml in a total of 100 μl and incubated at 37° C. for 60 minutes.Peroxidase-avidin at a concentration of 1 μl/ml in a total volume of 100μl was added and incubated at 37° C. for 30 minutes. The color reactionwas performed by adding to each well 100 μl of freshly preparedsubstrate solution and 0.03% H₂O₂ in 0.1 M sodium citrate (pH 4.3) andincubating the mixture at room temperature for 30 minutes. The plateswere read at 405 nm with a plate reader.

For 37 case patients with ovarian cancers and for 100 control subjects(about 70% of all subjects) a CA 125 level had been determinedpreviously (from the same specimens) and was available for comparison.These measurements had been performed with the original CA 125radioimmunoassay from Centocor and the assays were not repeated for thisstudy.

Statistical Analysis

Univariate comparisons for quantitative variables between normal andcancer cell lines or between case and control sera were made usingStudent's t test. For analysis of serum levels, adjustment for potentialconfounding variables such as the subject's age, year of collection andwhether the specimen had undergone freezing and thawing was carried outusing general linear modeling. Logistic regression analysis was used todetermine the statistical significance of both prostasin and CA 125 as apredictor of case status. Paired Student's t test was used to comparethe change in postoperative prostasin levels from preoperative levels.Pearson correlation coefficients were calculated between CA 125 andprostasin. Because the distributions of prostasin and CA 125 were skewedpositively, log-transformed values were used in statistical tests.Analyses with a P value of 0.05 or less were considered to bestatistically significant. All statistical tests were two sided and allconfidence intervals are 95%.

II. Results

Microarray analysis of pooled RNA isolated from three normal HOSE celllines and from three ovarian cancer cell lines was performed. Thirtygenes with Cy3/Cy5 signal ratios ranging from 5 to 444 were identified,suggesting that these genes were overexpressed in ovarian cancer cellscompared with normal HOSE cells. Among them, both prostasin andosteopontin encode secretory proteins which may be potential serummarkers. Another gene, creatine kinase B has been shown to produce aserum marker associated with renal carcinoma and lung cancer. Prostasinwas selected for further study because this gene had an availableantibody assay.

To evaluate the differential expression of prostasin in individualnormal and malignant ovarian epithelial cell lines derived from normaland neoplastic ovaries, we performed quantitative PCR analysis on fournormal HOSE cultures and on ten ovarian cancer cell lines. The relativeprostasin gene expression ranged from 120.3-fold to 410.1-fold greaterfor seven of the ten ovarian cancer cell lines compared with that forHOSE 697 cells but was only marginally greater for three other ovariancell lines. Overall, there was a highly statistically significantdifference between expression for the four normal cell lines compared tothe ten ovarian cancer cell lines P<0.001.

For further validation of the expression of prostasin in actual tumortissue, sections from two normal ovaries, from two serous borderlineovarian tumors and from two grade 1, two grade 2 and two grade 3 serousovarian cystadenocarcinomas were immunostained with an anti-prostasinpolyclonal antibody. Stronger cytoplasmic staining was detected incancer cells than in normal HOSE cells, suggesting that prostasin isoverexpressed by the ovarian cancer cells. Prostasin was, however, alsodetected in normal ovarian tissue by immunostaining. We next examinedprostasin levels detected by ELISA in sera from case patients andcontrol subjects. The mean prostasin level for all of the case patientswas 13.7 μg/ml compared with 7.5 μg/ml in all of the control subjects.Based on log-transformed values, this difference was statisticallysignificant (P<0.001) and persisted after adjustment for the subject'sage, year of collection, and quality of specimen (possible freeze-thawdamage). Among case patients, there was considerable variability bystage; however, notably-women with stage II disease had the highestlevels of prostasin, suggesting that prostasin may be of use forearly-stage detection. It also appeared that women with mucinous-typeovarian tumors had lower levels of prostasin than women with ovariantumors of other epithelial types. Among control subjects, there was astatistically significant tendency for the archived specimens to havelower prostasin levels than the current specimens (P<0.001), but therewas no evidence for an effect of age or diagnostic category (i.e.,normal tissue, benign gynecologic disease, or other gynecologic cancer).In addition, 60.5% of the archived case specimens and 66.2% of thecontrol specimens had been in the freezer in which freezing and thawinghad occurred. There was no evidence of a tendency for these samples tohave lower prostasin levels.

In sixteen women with nonmucinous epithelial ovarian cancers,preoperative and postoperative specimens were available for comparison.For fourteen of these women, a decreased prostasin level was observedafter surgery, and, in the entire group of sixteen, postoperative Plevels were statistically lower when compared with preoperative levelsusing a pair T test on the log-transformed values (P=0.004).

A bivariate plot of prostasin versus CA 125 performed for the 37 casepatients with nonmucinous ovarian cancers and for the 100 controlsubjects who had both measurements available failed to show astatistically significant correlation. This lack of correlation suggeststhat the two markers may provide complementary information. The combinedmarkers had a sensitivity of 34/37 (92%) and a specificity of 94/100(94%). In contrast, the sensitivity of CA 125 alone at the samespecificity was 24/37 (64.9%) and the sensitivity of prostasin alone atthe same specificity was 19/37 (51.4%).

III. Discussion

The present study demonstrates prostasin's potential as a biomarkerthrough real-time PCR in cancer and normal epithelial cell lines and bydifferential staining in cancer tissue compared with normal tissue.Higher levels of serum prostasin were found to be present in casepatients with ovarian cancer when compared to control subjects and adeclining level of prostasin was observed after surgery for ovariancancer. Results also suggest that assays with prostasin may be combinedwith those for other markers such as CA 125 to improve the reliabilityof procedures for the detection of ovarian cancer.

1. A method of determining whether a woman is at increased risk ofhaving ovarian cancer, comprising: a) obtaining a test sample containingovarian cells or tissue from said woman; b) determining the amount ofosteopontin protein in said test sample; c) comparing the results of thedetermination of step b) with results obtained using a control sample;and d) concluding that said woman is at increased risk of having ovariancancer if the amount of osteopontin in said test sample is higher thanthe amount in said control sample.
 2. The method of claim 1, whereinsaid amount of osteopontin protein is determined by means of an ELISAassay.
 3. The method of either of claims 1 or 2, further comprisingassaying said test sample for CA 125, comparing the results of thisdetermination with results obtained using a control sample; andconcluding that said woman is at increased risk of having ovarian cancerif the amount of CA125 in said test sample is higher than the amount insaid control sample.
 4. The method of either of claims 1 or 2, furthercomprising assaying said test sample for prostasin levels, comparing theresults of this determination with results obtained using a controlsample; and concluding that said woman is at increased risk of havingovarian cancer if the amount of prostasin in said test sample is higherthan the amount in said control sample.
 5. The method of either ofclaims 1 or 2, further comprising assaying said test sample for creatinekinase B levels, comparing the results of this determination withresults obtained using a control sample; and concluding that said womanis at increased risk of having ovarian cancer if the amount of creatinekinase B in said test sample is higher than the amount in said controlsample.